WO2007100009A1

WO2007100009A1 - Method for production of l-amino acid

Info

Publication number: WO2007100009A1
Application number: PCT/JP2007/053803
Authority: WO
Inventors: Yoshihiro Usuda; Kazuhiko Matsui
Original assignee: Ajinomoto Co., Inc.
Priority date: 2006-03-03
Filing date: 2007-02-28
Publication date: 2007-09-07
Also published as: EP2003209B1; EP2003209A1; US20090093029A1; US20140045228A1; JP2009118740A; EP2003209A4

Abstract

An L-amino acid can be produced by culturing a bacterium belonging to the family Enterobacteriaceae and capable of producing the L-amino acid in a culture medium containing glycerol, particularly crude glycerol, as a carbon source to produce and accumulate the L-amino acid in the culture and collecting the L-amino acid from the culture.

Description

Specification

L—Amino Acid Production Method

Technical field

[0001] The present invention relates to a method for producing an L-amino acid using a microorganism. L-amino acids are used in various fields such as seasonings, food additives, feed additives, chemical products, and pharmaceuticals. Background art

[0002] L-amino acids such as L-threonine and L-lysine are industrially produced by fermentation using L-amino acid-producing bacteria such as Escherichia bacteria having the ability to produce these L-amino acids. As these L-amino acid-producing bacteria, strains isolated from nature, human mutants of these strains, recombinants in which L-amino acid biosynthetic enzymes are enhanced by gene recombination, and the like are used. Examples of the method for producing L threonine include the methods described in Patent Documents:! On the other hand, examples of the method for producing L-lysine include the methods described in Patent Documents 5 to 8.

In the industrial production of L-amino acids by fermentation, sugars such as glucose, fructose, sucrose, molasses, and starch hydrolysates are used as carbon sources. Patent Document 1: JP-A-5-304969

Patent Document 2: Pamphlet of International Publication No. 98/04715

Patent Document 3: Japanese Patent Laid-Open No. 05-227977

Patent Document 4: US Patent Application Publication No. 2002/0110876

Patent Document 5: Japanese Patent Laid-Open No. 10-165180

Patent Document 6: Japanese Patent Laid-Open No. 11-92088

Patent Document 7: Japanese Unexamined Patent Publication No. 2000-253879

Patent Document 8: JP 2001-057896 A

Disclosure of the invention

Problems to be solved by the invention

[0003] In the present invention, a low-cost L-amino acid can be obtained by using a new raw material in the conventional fermentation method for L-amino acid using microorganisms that have been mainly performed using saccharides as a carbon source. The manufacturing method of this is provided.

Means for solving the problem

[0004] As a result of intensive studies to solve the above problems, the present inventors have cultivated bacteria belonging to the family Enterobacteriaceae and having L_amino acid-producing ability in a medium containing glycerol as a carbon source. It was found that L_amino acids can be produced in the same or more than a medium using sugar as a carbon source. Furthermore, as glycerol, it has been found that crude glycerol of low purity produced as a by-product in biodiesel fuel production, which is industrially produced worldwide, has a higher growth promoting effect than pure glycerol. The present invention has been completed based on the knowledge.

That is, the present invention is as follows.

(1) A bacterium belonging to the family Enterobacteriaceae and capable of producing L-amino acid is cultured in a medium containing glycerol as a carbon source, and L-amino acid is produced and accumulated in the culture, and L-amino acid is produced from the culture. A method for producing L-amino acids, characterized in that

(2) The said method whose initial concentration of glycerol in a culture medium is 1-30 w / v%.

(3) The method as described above, wherein the medium is a medium supplemented with crude glycerol.

(4) The method as described above, which is a crude glyceride produced by the production of the crude glycerol-powered biodiesel fuel.

(5) The crude glycerol power The method as described above, which is glycerol capable of producing more L-amino acids when used as a carbon source compared to the reagent glycerol.

(6) The method as described above, wherein the bacterium belongs to the genus Escherichia.

(7) The method as described above, wherein the bacterium belongs to the genus Pantoea.

(8) The method as described above, wherein the bacterium is Escherichia coli.

(9) The method as described above, wherein the L-amino acid is L-threonine or L-lysine.

(10) the L-amino acid is L-threonine, and the bacterium is aspartate semialdehyde dehydrogenase, aspartokinase I encoded by the thr operon, homoserine kinase, aspartate aminotransferase, and The method, wherein the activity of one or more enzymes selected from the group consisting of threonine synthase is enhanced.

(11) The L amino acid is L lysine, and the bacterium is dihydrodipicolinate reductor. Diaminopimelate decarboxylase, diaminopimelate dehydrogenase, phosphonolpyruvate carboxylase, aspartate aminotransferase, diaminopimelate epimerase, aspartate semialdehyde dehydrogenase, tetrahydrodipicolinate succinylase, and succinyl diamino The aforementioned method, wherein the activity of one or more enzymes selected from the group consisting of pimelate deacylase is enhanced and / or the activity of lysine decarboxylase is attenuated.

(12) The L_amino acid is L-gnoretamic acid, and the bacterium is selected from the group consisting of glutamate dehydrogenase, citrate synthase, phosphoenolpyruvate carboxylase, and methyl thioate synthase. The above-mentioned method, wherein the activity of the enzyme is enhanced and / or the activity of monoketoglutarate dehydrogenase is weakened.

(13) The L-amino acid is L-tryptophan, and the bacterium is phosphoglycerate dehydrogenase, 3-deoxy D-arabinohepturonic acid 7-phosphate synthase, 3-dehydroquinate synthase, shikimate dehydrogenase, shikimi The activity of one or more enzymes selected from the group consisting of acid kinase, 5-enolpyruvylshikimate 3-phosphate synthase, chorismate synthase, prefenate dehydratase, and chorismate mutase is enhanced. The above method.

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be described in detail.

<1> Glycerol used in the present invention

Glycerol refers to the substance with the official name Propane_l, 2,3-triol. In the present invention, crude glycerol refers to glycerol containing impurities produced industrially. Crude glycerol is industrially produced by hydrolyzing fats and oils in contact with water at high temperature and high pressure, or by esterification reaction for biodiesel fuel production. Biodiesel fuel is a fatty acid methyl ester produced by transesterification from fats and methanol, and crude glycerol is produced as a byproduct of this reaction (Fukuda, H., Kondo, A., and Noda, H. 2001, J. Biosci. Bioeng. 92, 405-416). In the biodiesel production process, transesterification is alkaline. Since an acid is added at the time of neutralization which is often used in the medium method, crude glycerol containing water and impurities and having a purity of about 70 to 95% by weight is produced. Crude glycerol produced in the production of biodiesel fuel contains, in addition to water, salts of residual methanol and catalysts such as NaOH such as NaOH and acids such as KSO used for neutralization. Manufacturer

twenty four

The power varies depending on the manufacturing method. Such salts and methanol reach several percent. Here, ions derived from alkali or acid used for neutralization thereof such as sodium, potassium, chloride ion and sulfate ion are 2 to 7%, preferably 3 to 6% based on the weight of crude glycerol. %, More preferably 4 to 5.8% preferred. The methanol is contained as an impurity, it is not necessary, but it is preferably contained less than 0.01%.

[0007] Furthermore, the crude glycerol may contain trace amounts of metals, organic acids, phosphorus, fatty acids and the like. Examples of the organic acid included include formic acid and acetic acid. The organic acid may not be included as an impurity, but preferably 0.01% or less. Examples of trace metals contained in the crude glycerol include magnesium, iron, calcium, manganese, copper, and zinc, which are preferred for the growth of microorganisms. Magnesium, iron, and calcium may be contained in a total amount of 0.00001 to 0.1%, preferably 0.0005 to 0.1%, more preferably 0.004 to 0.05%, and still more preferably 0.007 to 0.01%, based on the weight of the crude glycerol. I like it. As manganese, copper and zinc, the total content is preferably 0.000005 to 0.01%, more preferably 0.000007 to 0.005%, and still more preferably 0.00001 to 0.001%.

[0008] The purity of the crude glycerol is preferably 10% or more, more preferably 50% or more, further preferably 70% or more, and particularly preferably 80% or more. As long as the impurity content satisfies the above range, the purity of glycerol may be 90% or more.

[0009] A preferred crude glycerol in the present invention is a crude glycerol produced in the production of biodiesel fuel. The preferred crude glycerol in the present invention means glycerol capable of producing more L-amino acids when used as a carbon source compared to the same amount of reagent glycerol. Producing more L_amino acids compared to reagent glycerol compared to using reagent glycerol as a carbon source. L means an increase in amino acid production capacity%, preferably 10%, more preferably 20% or more. “Reagent glycerol” means glycerol which is commercially available as a so-called reagent grade or glycerol having an equivalent purity, and it is particularly preferable that the purity is 99% by weight or more, and pure glycerol is particularly preferable. “The same amount of reagent glycerol as crude glycerol” means that when the crude glycerol contains water, the amount of reagent glycerol is the same as the remaining weight excluding water.

[0010] In the present invention, the crude glycerol may be diluted with a solvent such as water. In that case, the above description regarding the content of glycerol and impurities applies to the crude glycerol before dilution. That is, when the crude glycerol contains a solvent such as water, the solvent is removed so that the solvent content is preferably 30% by weight or less, more preferably 20% by weight or less, and even more preferably 10% by weight or less. In addition, if the content range of glycerol and impurities is satisfied, it corresponds to “crude glycerol” in the present invention.

[0011] <2> Bacteria used in the present invention

In the present invention, a bacterium belonging to the family Enterobacteriaceae and having an L amino acid-producing ability is used.

Enterobacteriaceae include bacteria belonging to genera such as Escherichia, Enteropactor, Ervinia, Klebsiella, Pantoea, Photorha grapes, Providencia, Salmonella, Serratia, Shigura, Morganella, Yersinia and others. In particular, NCBI (National Center for Biotechn ology Information) § ¹ ~~ eat ¹ ~ ~ vinegar: at (http //www.ncbi.nlm mh.gov/Taxonomy/Browser /wwwtax.cgi?id=91347.) Bacteria classified into the family Enterobacteriaceae according to the classification method used are preferred.

[0012] The bacterium belonging to the genus Escherichia is not particularly limited, but means that the bacterium is classified into the genus Escherichia according to a classification known to a specialist in microbiology. Examples of bacteria belonging to the genus Escherichia used in the present invention include, but are not limited to, Escherichia coli (E. coli).

[0013] The bacteria belonging to the genus Escherichia that can be used in the present invention are not particularly limited. For example, Neidhardt, FC Ed. 1996. Escherichia coll and salmonella: ellular and Molecular Biology / Second Edition pp. 2477-2483. Ta ble 1. Includes those described in the American Society for Microbiology Press, Washington, DC. Specific examples include Escherichia coli W3110 (ATCC 27325) and Escherichia coli MG1655 (ATCC 47076) derived from the prototype wild type K12 strain.

[0014] These strains are, for example, American type culture 'collection (address P.O. Box)

1549 Manassas, VA 20108, United States of America). In other words, a registration number corresponding to each strain is given, and it is possible to receive sales by using this registration number. The registration number corresponding to each strain is listed in the catalog of the American 'type' culture collection.

Bacteria belonging to the genus Pantoea means that the bacterium is classified as belonging to the genus Pantoea according to the classification known to microbiologists. Certain types of enteropactor 'agglomerans have recently been reclassified into Pantoea's Glomerance, Pantoea's Ananatis, Pantoea's Stealty, etc. (Int. J. Syst. Bacteriol ., 43, 162-173 (1993)). In the present invention, the bacteria belonging to the genus Pantoea include bacteria that have been reclassified to the genus Pantoea in this way.

[0015] The bacterium used in the present invention has a reduced expression of glpR gene (EP17 15056), glpA, glpB, glpC, glpD, glpE, glpF, glpG, glpK, Expression of glycerol metabolism genes (EP1715055A) such as glpQ, glpT, glpX, tpiA, gldA, dhaK ゝ dhaL, dhaM ゝ dhaR ゝ fsa and ta gene may be enhanced.

[0016] In the present invention, the bacterium having an amino acid-producing ability refers to a bacterium having an ability to produce L-amino acid and secrete it into the medium when cultured in the medium. Preferably, it refers to a bacterium capable of accumulating the target L-amino acid in the medium in an amount of preferably 0.5 g / L or more, more preferably 1.0 g / L or more. L-amino acids include L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cystine, L-glutamic acid, L-glutamine, glycine, L-histidine, L-isoleucine, L-leucine, L- Contains lysine, L_methionine, L-phenenolealanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine and L-valine. In particular, L-threonine, L-lysine and L-glutamic acid are preferred. Hereinafter, a method for imparting L amino acid-producing ability to the bacterium as described above or a method for enhancing the bacterial L-amino acid producing ability as described above will be described.

[0017] To impart L-amino acid-producing ability, the acquisition of auxotrophic mutants, L-amino acid analog-resistant strains or metabolically controlled mutants, and the expression of L_amino acid biosynthetic enzymes were enhanced. Conventional methods such as the creation of recombinant strains that have been adopted for breeding amino acid-producing bacteria such as coryneform bacteria or bacteria belonging to the genus Escherichia can be applied (Amino Acid Fermentation, Japan Publishing Center, Inc., 1986 5). 30th month, first edition, 77-: 100 pages). Here, in the breeding of L-amino acid-producing bacteria, the auxotrophy, analog resistance, metabolic control mutation, and other properties imparted may be single or two or more. In addition, L_ amino acid biosynthetic enzymes whose expression is enhanced may be used alone or in combination of two or more. Furthermore, imparting properties such as auxotrophy, analog resistance, and metabolic regulation mutation may be combined with enhancement of biosynthetic enzymes.

[0018] To obtain an auxotrophic mutant, an analog-resistant strain, or a metabolically controlled mutant having L amino acid-producing ability, the parent strain or wild strain is subjected to normal mutation treatment, that is, irradiation with X-rays or ultraviolet rays, or N Methyl-N, 12 Throot N Treated with a treatment with a mutant such as ditrosoguanidine, etc., and shows auxotrophy, analog resistance, or metabolically controlled mutations among the mutants obtained, and produces L amino acids You can get the power S by selecting the ones that have the ability.

[0019] The L amino acid-producing ability can also be imparted or enhanced by enhancing enzyme activity by gene recombination. Examples of the enhancement of enzyme activity include a method of modifying a bacterium so that expression of a gene encoding an enzyme involved in L amino acid biosynthesis is enhanced. As a method for enhancing the expression of a gene, an amplified plasmid in which a DNA fragment containing the gene is introduced into an appropriate plasmid, for example, a plasmid vector containing at least a gene responsible for the replication and replication function of the plasmid in a microorganism, is introduced. Alternatively, these genes can be achieved by making multiple copies on the chromosome by joining, transferring, etc., or introducing mutations into the promoter region of these genes (International Publication Pamphlet W095 / 34672). reference).

[0020] When the target gene is introduced onto the amplification plasmid or chromosome, these genes Any promoter may be used so long as it functions in coryneform bacteria, and it may be the promoter of the gene itself to be used or a modified one. The expression level of a gene can also be regulated by appropriately selecting a promoter that functions strongly in coryneform bacteria, or by bringing the 35th and 10th regions of the promoter closer to the consensus sequence. The method for enhancing the expression of the enzyme gene as described above is described in WO00 / 18935 pamphlet, European Patent Application Publication No. 1010755, and the like.

[0021] Hereinafter, a method for imparting L-amino acid-producing ability to bacteria and a bacterium imparted with L-amino acid-producing ability will be exemplified.

[0022] L-threonine-producing bacteria

Preferable microorganisms having L-threonine-producing ability include bacteria with enhanced activity of one or more L-threonine biosynthesis enzymes. L-threonine biosynthesis enzymes include aspartokinase III (lysC), aspartate semialdehyde dehydrogenase ( _as d), aspartokinase I (thrA) encoded by the thr operon, and homoserine kinase ( thrB), threonine synthase (thrC), and aspartate aminotransferase (aspartrate transaminase) (aspC). The inside of Katsuko is an abbreviation of the gene (the same applies to the following description). Among these enzymes, aspartate semialdehyde dehydrogenase, aspartokinase I, homoserine kinase, aspartate aminotransferase, and threonine synthase are particularly preferred.

The L-threonine biosynthesis gene may be introduced into a bacterium belonging to the genus Escherichia in which threonine degradation is suppressed. Examples of bacteria belonging to the genus Escherichia in which threonine degradation is suppressed include the TDH6 strain lacking threonine dehydrogenase activity (Japanese Patent Laid-Open No. 2001-346578).

[0023] The enzymatic activity of the L-threonine biosynthetic enzyme is suppressed by the final product, L-threonine. Therefore, in order to construct an L-threonine-producing bacterium, it is desirable to modify the L-threonine biosynthetic gene so that it is not subject to feedback inhibition by L-threonine. The thrA, thrB, and thrC genes above constitute the threonine operon. The threonine operon forms an attenuator structure, and the threonine operon Expression is inhibited by isoleucine and threonine in the culture medium, and expression is suppressed by attenuation. This modification can be achieved by removing the leader region or the attenuation region of the attenuation region. (Lynn, SP, Burton, WS, Donohue, TJ, Gould, RM, Gumport, RI, and Gardner, JFJ Mol. Biol. 194: 59-69 (1987); WO 02/26993 pamphlet; 2005/049808 No. Pan frets, Teru)

[0024] Upstream of the threonine operon, a unique promoter S may be substituted with a non-natural promoter (see WO98 / 04715 pamphlet), or the expression of a gene involved in threonine biosynthesis is expressed by lambda phage. A threonine operon may be constructed that is governed by the repressor and promoter. (See European Patent No. 0593792) In order to modify bacteria so that it is not subject to feedback inhibition by L-threonine, a strain resistant to a-amino-β-hydroxyvaleric acid (AHV) may be selected. It is possible.

[0025] The threonine operon that has been modified in such a way that it is not subject to feedback inhibition by L-threonine is linked to a force that increases the copy number in the host or to a strong promoter, and the expression level is It is preferable to improve. The increase in copy number can be achieved by transferring the threonine operon on the genome by transposon, Mu-phage, etc. in addition to amplification by plasmid.

[0026] In addition to the L-threonine biosynthetic enzyme, it is also preferable to enhance the genes involved in glycolysis, TCA cycle, respiratory chain, genes controlling gene expression, and sugar uptake genes. These L-threonine-producing genes include transhydronase (pntAB) gene (European Patent 733712), phosphoenolpyruvate carboxylase gene (P mark C) (International Publication No. 95/06114 Pamphlet) ), Phosphoenolpyruvate synthase gene (pps) (European Patent No. 877090), pyruvate carboxylase gene of coryneform or Bacillus genus (International Publication No. 99/18228, European Application Publication No. 1092776) Is mentioned.

[0027] In addition, the expression of a gene conferring resistance to L-threonine, the gene conferring resistance to L-homoserine, and the addition of L-threonine resistance and L-homoserine resistance to the host are added. It is also suitable to give. Examples of genes that confer resistance include rhtA gene (Res. Microbio 1. 154: 123-135 (2003)), rhtB gene (European Patent Application Publication No. 0994190), rht C gene (European Patent Application Publication No. 1013765). No. description), yfiK, yeaS gene (European Patent Application Publication No. 1016710). For methods for imparting L-threonine resistance to a host, methods described in European Patent Application Publication No. 0994190 and International Publication No. 90/04636 can be referred to.

[0028] Examples of L-threonine-producing bacteria or parent strains for deriving them include E. coli TDH-6 / pVIC40 (VKPM B-3996) (US Pat. No. 5,175,107, US Pat. , 705,371), E. coli 472T23 / pYN7 (ATCC 98081) (US Pat. No. 5,631,157), E. coli NRRL-21593 (US Pat. No. 5,939,307), E. coli FERM BP -3756 (US Pat. No. 5,474,918), E. coli FERM BP-3519 and FERM BP-3520 (US Pat. No. 5,376,538), E. coli MG442 (Gu syatiner et al, Genetika (in Russian) ), 14, 947-956 (1978)), E. coli VL643 and VL2055 (EP 1149911 A), and other strengths including, but not limited to, strains belonging to the genus Escherichia.

[0029] The TDH-6 strain lacks the thrC gene, is sucrose-assimilating, and has an ilvA gene force S leaky mutation. This strain also has a mutation in the rhtA gene that confers resistance to high concentrations of threonine or homoserine. The strain B-3996, the RSF1010-derived vector, which contains the plasmid pVIC40 which was obtained by inserting a thrA * BC operon which includes a mutant _t hrA gene. This mutant thrA gene encodes aspartokinase homoserine dehydrogenase I which is substantially desensitized to feedback inhibition by threonine. The B-3996 stock was deposited on 19 November 1987 with the accession number RIA 1867 in the all-union 'Scientific' Center 1 'Ob' Antibiotics (Nagatinskaya Street 3_A, 117105 Moscow, Russia). This strain was also transferred to Lucian 'National' Collection 'Ob' Industrial 'Microorganisms (VKPM) (1 Dorozhny proezd., 1 Moscow 117545, Russia) on April 7, 1987. Deposited at 3996.

[0030] E. coli VKPM B-5318 (EP 0593792B) can also be used as an L_threonine-producing bacterium or a parent strain for inducing it. B-5318 is an isoleucine non-requirement, and the control region force of the threonine operon in plasmid PVIC40 is temperature sensitive lambda phage C1 repressor. And the PR promoter. VKPM B-5318 was assigned to Lucian 'National' Collection 'Ob Industrial Microorganisms (V KPM) (1 Dorozhny proezd., 1 Moscow 117545, Russia) on May 3, 1990. It is deposited internationally at -5318.

[0031] The thrA gene encoding aspartokinase homoserine dehydrogenase I of Escherichia coli has been elucidated (nucleotide numbers 337 to 2799, GenBank accession NC — 0 00913.2, gi: 49175990). The thrA gene is located between the thrL gene and the thrB gene in the chromosome of E. coli K-12. The thrB gene encoding homoserine kinase of Escherichia coli has been elucidated (nucleotide numbers 2801 to 3733, GenBank accessi on NC_000913.2, gi: 49175990). The thrB gene is located between the thrA gene and the thrC gene in the chromosome of E. coli K-12. The thrC gene encoding the threonine synthase of Escherichia coli has been elucidated (nucleotide numbers 3734-5020, GenBank accession NC— 000913.2, gi: 49175990). The thrC gene is located between the thrB gene and the yaaX open reading frame in the chromosome of E. coli K-12. All three of these genes function as a single threonine operon. To increase the expression of the threonine operon, the attenuator region that affects transcription is preferably removed from the operon (WO2005 / 049808, WO2003 / 097839).

[0032] Mutant thrA encoding aspartokinase homoserine dehydrogenase I resistant to feedback inhibition by threonine, and thrB gene and thrC gene are known plasmids present in threonine-producing strain E. coli VKPM B-3996 It can be acquired as one operon from pVIC40. Details of plasmid pVIC40 are described in US Pat. No. 5,705,371.

[0033] The rhtA gene is present on the 18th minute of the Coli chromosome near the glnHPQ operon, which encodes an element of the glutamine transport system. The rhtA gene is identical to ORF1 (ybiF gene, nucleotide number 764-16541, GenBank accession number AAA218541, gi: 440181), and is located between the pexB gene and the ompX gene. The unit that expresses the protein encoded by ORF1 is called the rhtA gene (rht: resistant to homoserine and threonine). It was also found that the rhtA23 mutation is a G → A substitution at position -1 relative to the ATG start codon. ABSTRACTS of the 17th International Congress of Biochemistry and Molecular Biology in conjugation with Annual Meeting of the American Society for Biochemistry and Molecular Biology, San Francisco, California August 24-29, 1997, abstract No. 457, EP 1013765 A ).

[0034] The asd gene of E. coli has already been clarified (nucleotide numbers 3572511 to 3571408, GenBank accession NC_000913.1, gi: 16131307), and by PCR using a primer prepared based on the base sequence of the gene. (See White, TJ et al., Tren ds Genet., 5, 185 (1989)). Other microbial asd genes can be obtained as well.

[0035] In addition, the aspC gene of E. coli has already been clarified (nucleotide numbers 983742 to 984932, GenBank accession NC — 000913.1, gi: 16128895) and can be obtained by PCR. The aspC gene of other microorganisms can be obtained similarly.

[0036] L-lysine-producing bacteria

Examples of L-lysine-producing bacteria belonging to the genus Escherichia include mutants having resistance to L-lysine analogues. L-lysine analogues inhibit the growth of bacteria belonging to the genus Escherichia, but this inhibition is completely or partially desensitized when L-lysine is present in the medium. Examples of L lysine analogues include, but are not limited to, oxalidine, lysine hydroxamate, S- (2-aminoethyl) L cysteine (AEC), y-methyl lysine, α-chlorocaprolatatam, and so on. Les. Mutants having resistance to these lysine analogs can be obtained by subjecting bacteria belonging to the genus Escherichia to normal artificial mutation treatment. Specific examples of bacterial strains useful for the production of L-lysine include Escherichia coli AJ11442 (FERM BP-1543, NRRL B-12185; see US Pat. No. 4,346,170) and Escherichia coli VL611. In these microorganisms, feedback inhibition of aspanolet kinase by L-lysine is released.

[0037] The WC196 strain can be used as an L-lysine-producing bacterium of Escherichia coli. This strain was bred by conferring AEC resistance to the W3110 strain derived from Escherichia coli K-12. This strain was named Escherichia coli AJ13069.December 6, 1994, National Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology (currently the National Institute of Advanced Industrial Science and Technology (AIST), τ 305-8566 Japan Ibaraki Tsukuba Sakai Higashi 1-chome 1 1 Central 6) Contract number FER Deposited as MP-14690, transferred to an international deposit under the Budapest Treaty on September 29, 1995, and assigned the accession number FERM BP-5252 (US Pat. No. 5,827,698).

[0038] Examples of L-lysine-producing bacteria or parent strains for deriving the same also include strains in which one or more activities of L-lysine biosynthesis enzyme are enhanced. Examples of such enzymes include dihydrodipicolinate synthase (dapA), aspartokinase (lysC), dihydrodipicolinate reductase (dapB), diaminopimelate decarboxylase (lysA), diaminopimelate dehydrogenase (ddh) (US patent) 6,040, 160), phosphoenolpyruvate carboxylase (ppc), aspartate semialdehyde dehydrogenase gene, diaminopimelate epimerase (dapF), tetrahydrodipicolinate succinylase (dap D), succinyl diaminopimelate Powers including, but not limited to, deacylase (dapE) and aspartase (aspA) (EP 1 253195 A). Among these enzymes, dihydrodipicolinate reductase, diaminopimelate decarboxylase, diaminopimelate dehydrogenase, phosphoenolpyruvate carboxylase, aspartate aminotransferase, diaminopimelate epimerase, aspartate semialdehyde dehydrogenase, tetrahydrodipicolinate succinylase, And succinyl diaminovimelic acid deacylase is particularly preferred. Further, the parent strain is a gene involved in energy efficiency (cyo) (EP 1170376 A), a gene encoding nicotinamide nucleotide transhydrogenase (pntAB) (US Pat.No. 5,830,716), ybjE gene (WO2005 / 073390), Or, the expression level of these combinations increases.

[0039] Examples of L-lysine-producing bacteria or parent strains for deriving them include reduced or deficient activity of enzymes that catalyze reactions that branch off from the L-lysine biosynthesis pathway to produce compounds other than L-lysine. The stock which is doing is also mentioned. Examples of enzymes that catalyze reactions that branch off from the biosynthetic pathway of L_lysine to produce compounds other than L—lysine include homoserine dehydrogenase, lysine decarboxylase (US Pat. No. 5,827,698), and malate enzyme ( WO2005 / 010175).

[0040] Preferred L-lysine-producing bacteria include Escherichia coli WC196DcadADldc / pCABD2 (WO2006 / 078039). This strain is a WC196 strain in which the cad A and ldcC genes encoding lysine decarboxylase are disrupted, and the strain described in US Pat. No. 6,040,160. Smid is a strain obtained by introduction of pCABD2. PCABD2 is a mutant dapA gene encoding dihydrodipicolinate synthase (DDPS) derived from Escherichia coli having a mutation that has been desensitized to feedback inhibition by L-lysine, and feedback inhibition by L-lysine has been eliminated. Mutant lysC gene encoding aspartokinase III derived from Escherichia coli with mutation, dapB gene encoding dihydrodipicolinate reductase derived from Escherichia coli, and Brevibaterium 'ratatofarmentum It contains the ddh gene encoding the derived diaminobimelate dehydrogenase. Escherichia coli W3110 (tyrA) / pCABD2 carrying this plasmid was named AJ12604, and on January 28, 1991, National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center (address τ 305-8566, Ibaraki, Japan) Deposited with FERM P-11975 deposit number at Tsukuba Rakuto 1-chome 1 1-center 6), transferred to international deposit under the Budapest Treaty on September 26, 1991, and FERM BP-35 79 Deposited by number.

[0041] L-cysteine producing bacteria

Examples of L-cystine producing bacteria or parent strains for inducing them include E. coli JM15 (US Pat. No. 6,218,168, Russia) transformed with a different cysE allele encoding a serine acetyltransferase resistant to feedback inhibition. (Patent Application No. 2003121601), E. coli W3110 (US Pat.No. 5,972,663) with an overexpressed gene encoding a protein suitable for excretion of toxic substances into cells, decreased cysteine desulfhydrase activity E. coli strains (JP11155571A2), E. coli W3110 (WO0127307A1) and other strains belonging to the genus Escherichia with increased activity of the transcriptional regulator of the positive cystine regulon encoded by the cysB gene. Not.

[0042] L_leucine producing bacteria

Examples of L leucine-producing bacteria or parent strains for inducing the same include leucine-resistant E • coil strains (eg, 57 strains (VKPM B-7386, US Pat. No. 6,124,121)) or j3 — 2 —E.coli strains resistant to leucine analogs such as phenylalanine, 3-hydroxyleucine, 4-azaleucine, 5, 5, 5-trifluoroleucine (JP-B-62-34397 and JP-A-8-70879), WO96 Strains belonging to the genus Escherichia such as E. coli strains and E. coli H-9068 (JP-A-8-70879) obtained by the genetic engineering method described in / 06926, but are not limited thereto. I can't.

[0043] The bacterium used in the present invention may be improved by increasing the expression of one or more genes involved in L-mouth isin biosynthesis. As an example of such a gene, a leuABCD operon represented by a mutant leuA gene (US Pat. No. 6,403,342) encoding isopropyl malate synthase, which is preferably desensitized to feedback inhibition by L-leucine, is used. Gene. Furthermore, the bacterium used in the present invention may be improved by increasing the expression of one or more genes encoding proteins that excrete L_ amino acids from bacterial cells. Examples of such genes include b2682 gene and b2683 gene (ygaZH gene) (EP 1239041 A2).

[0044] L_histidine-producing bacteria

Examples of L-histidine-producing bacteria or parent strains for inducing them include E. coli 24 strain (VKPM B-5945, RU2003677), E. coli 80 strain (VKPM B-7270, RU2119536), E. coli NR RL B-12116-B12121 (US Patent No. 4,388,405), E. coli H-9342 (FERM BP-6675) and H-9343 (FERM BP-6676) (US Patent No. 6,344,347), E. coli H-9341 (FERM B P-6674) (EP1085087), E. coli AI80 / pFM201 (US Pat. No. 6,258,554) and other forces S, including but not limited to strains belonging to the genus Escherichia.

[0045] Examples of L-histidine-producing bacteria or parent strains for deriving the same also include strains in which expression of one or more genes encoding L-histidine biosynthetic enzymes are increased. Examples of such genes include the ATP phosphoribosyltransferase gene (hisG), the phosphorifosyl AMP cyclohydrolase gene (hisl), the phosphorifosyl-ATP pyrophosphohydrolase gene (hisl), and the phosphorifosylformimimino. -5-Aminoimidazole carboxamide ribotide isomerase gene (hisA), amide transferase gene (his H), histidinol phosphate aminotransferase gene (hisC), histidinol phosphatase gene (hisB), histidinol dehydrogenase gene (hisD) And so on.

[0046] L-histidine biosynthetic enzymes encoded by hisG and hisBHAFI are known to be inhibited by L-histidine. Therefore, L-histidine-producing ability is determined by the ATP phosphoryltransferase gene (hisG Mutations that confer resistance to feedback inhibition Can be efficiently increased (Russian Patent Nos. 2003677 and 2119536).

[0047] Specific examples of strains having the ability to produce L histidine include E. coli FERM-P 5038 and 5048 into which a vector carrying a DNA encoding an L histidine biosynthetic enzyme has been introduced (Japanese Patent Laid-Open No. 56-005099). ), E. coli strain into which an amino acid transport gene has been introduced (EP1016710A), E. coli strain 80 with resistance to sulfaguanidine, DL-1,2,4-triazole-3-alanine and streptomycin (VKPM B-7270) , Russian Patent No. 2119536).

[0048] L-glutamic acid-producing bacterium

Examples of L-gnoretamic acid-producing bacteria or parent strains for deriving the same include, but are not limited to, strains belonging to the genus Escherichia such as E. coli VL33 4thrC ⁺ (EP 1172433). E. coli VL334 (VKPM B-1641) is an L-isoleucine and L-threonine-requiring strain having mutations in the thrC gene and the ilvA gene (US Pat. No. 4,278,765). The wild type allele of the thrC gene was introduced by a general transduction method using butteriophage P1 grown in cells of the wild type E. coli K12 strain (VKPM B_7). As a result, L-isoleucine-requiring L-glutamic acid-producing bacterium VL334thrC ⁺ (VKPM B-8961) was obtained.

[0049] Examples of L-gnoretamic acid-producing bacteria or parent strains for deriving the same include, but are not limited to, a strain with enhanced activity of one or more L-dalamic acid biosynthetic enzymes. . Examples of such genes include glutamate dehydrogenase (gdhA), glutamine synthetase (glnA), glutamate synthetase (gltAB), isocitrate dehydrogenase (icdA), aconate hydratase (acnA, acnB), citrate synthase (gltA) ), Methyl citrate sinter (p 卬 C), phosphoenol pyruvate carbocilase (ppc), pyruvate dehydrogenase (aceEF, lpdA), pyruvate kinase (pykA, pykF), phosphoenol pyruvate Synthase (pp _S A), enolase (eno), phosphoglyceromutase (pgmA, pgml), phosphoglycerate kinase (pgk), glyceraldehyde-3-phosphate dehydrogenase (gapA), triphosphate Fate isomerase (tpiA), fructose bisphosphate aldolase (ftp), phosphoflux Tokinase (pfkA, pikB), glucose phosphate isomerase (pgi) and the like. Among these enzymes, Glutamate dehydrogenase, citrate synthase, phosphonolpyruvate carboxylase, and methyl citrate synthase are preferred.

[0050] Examples of strains that have been modified to increase expression of the citrate synthetase gene, the phosphoenolpyruvate carboxylase gene, and / or the gnoretamate dehydrogenase gene include EP1078989A, EP955368A, and EP952221A. Those disclosed in (1).

[0051] Examples of L-gnoretamic acid-producing bacteria or parent strains for deriving them include activity of enzymes that catalyze the synthesis of compounds other than L-glutamic acid by branching from the biosynthetic pathway of L-daltamic acid. Also included are strains that are reduced or deficient. Examples of such enzymes include isocitrate lyase (aceA), hypoketoglutarate dehydrogenase (sucA), phosphoto lance acetylase (pta), acetate kinase (ack), acetate hydroxy acid synthase (ilv G), acetate lactate synthase (ΠνΙ), formate acetyl transferase (pfl), lactate dehydrogenase (ldh), glutamate decarboxylase (gadAB) and the like. Bacteria belonging to the genus Escherichia lacking α-ketoglutarate dehydrogenase activity or having reduced α-ketoglutarate dehydrogenase activity, and methods for obtaining them are described in US Pat. Nos. 5,378,616 and 5,573,945. RU

[0052] Specific examples include the following.

E. coli W3110sucA :: Kmr

E. coli AJ12624 (FERM BP- 3853)

E. coli AJ12628 (FERM BP-3854)

E. coli AJ 12949 (FERM BP- 4881)

[0053] E. coli W3110sucA :: Kmr is a strain obtained by disrupting the E- coli W3110 hypoketoglutarate dehydrogenase gene (hereinafter also referred to as "sucA gene"). This strain is completely deficient in α-ketognoletalate dehydrogenase.

[0054] Other examples of L-gnoretamic acid-producing bacteria include those belonging to the genus Escherichia and having resistance to aspartic acid antagonists. These strains may be deficient in hyketoglutarate dehydrogenase, for example, E. coli AJ13199 (FERM BP-5807) (US Pat. No. 5,908,768), and L-gnoretamic acid FFRM P_12379 (US (National Patent No. 5,393,671); AJ13138 (FERM BP-5565) (US Patent No. 6,110,714).

An example of an L-glutamic acid-producing bacterium of Pantoea ananatis is Pantoea anana tes AJ13355 strain. This strain was isolated from the soil of Kamata Pass in Shizuoka Prefecture as a strain that can grow on a medium containing L-gnoretamic acid and a carbon source at low pH. On February 19, 1998, Panto-Ananatis AJ13355 was registered as a patent biological deposit center of the National Institute of Advanced Industrial Science and Technology. Was deposited under the accession number FERM P-16644, transferred to an international deposit under the Budapest Treaty on 11 January 1999, and given the accession number FERM BP-6614. The strain was identified as Enterobacter ag glomerans at the time of its isolation, and was deposited as Enterobacter agglomerans AJ13355. anana tis).

[0056] Further, L-gnoretamic acid-producing bacteria of Panto'ananatis include bacteria belonging to the genus Pantoea in which α-ketognoletaleate dehydrogenase (a KGDH) activity is deficient or a KGDH activity is reduced. Such strains include AJ13356 (US Pat. No. 6,331,419) in which the ct KGDH-E 1 subunit gene ( _SUC A) of AJ13355 strain is deleted, and SC17 selected as a mucus low-producing mutant strain from AJ13355 strain. There is SC17sucA (US Pat. No. 6,596,517) which is a sucA gene deficient strain derived from the strain. AJ13356 was founded on February 19, 1998, National Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology (Currently the National Institute of Advanced Industrial Science and Technology (AIST), Biological Depositary Center, τ 305-8566 1) Deposited under FERM P-16645 at No. 6), transferred to an international deposit under the Budapest Treaty on 11 January 1999, and given the FERM BP-6616. AJ13355 and AJ13356 are deposited as Enterobacter agglomerans in the above depository organization, but are described as Pantoea ananatis in this specification. The SC17sucA strain was assigned the private number AJ417 and was deposited at the National Institute of Advanced Industrial Science and Technology as the Patent Biological Deposit Center on February 26, 2004 under the accession number FERM BP-08646.

[0057] Furthermore, as an L-glutamic acid-producing bacterium of Pantoea ananatis, SC17sucA / RSFC PG + pSTVCB strain, AJ13601 strain, NP106 strain, and NA1 strain. SC17sucA / RSFC PG + pSTVCB strain is a plasmid RSFCPG containing the citrate synthase gene (gltA), the phosphoenolpyruvate carboxylase gene (ppsA), and the glutamate dehydrogenase gene (gdhA) derived from Escherichia coli. In addition, it is a strain obtained by introducing a plasmid pSTVCB containing a citrate synthase gene (gltA) derived from Brevibaterium ratatofamentum. The AJ13601 strain was selected from the SC17sucA / RSFCPG + pSTVC B strain as a strain resistant to high concentrations of L-gnoretamic acid at low pH. The NP106 strain is a strain obtained by removing the plasmid RSFCPG + pSTVCB from the AJ13601 strain as described in the Examples. On August 18, 1999, AJ13601 shares were registered with the Patent Biological Depositary Center of the National Institute of Advanced Industrial Science and Technology (T 305-8566, Ibaraki, Tsukuba, 1-chome, 1-Chuo 6). Deposited as P-17516, transferred to an international deposit based on the Budapest Treaty on July 6, 2000, and assigned accession number FERM BP-7207.

L—Phenilalanin producing bacteria

Examples of L-phenylalanine-producing bacteria or parent strains for inducing them include E.coli AJ12739 (tyrA :: TnlO, tyrR) (VKPM) deficient in chorismate mutase-prefenate dehydrogenase and tyrosine repressor. B_8197) (WO03 / 044191), E. coli HW1089 (ATCC 55371) carrying a mutant phe A34 gene encoding chorismate mutase-prefenate dehydratase with desensitized feedback inhibition (US Pat.No. 5,354,672) Strains belonging to the genus Escherichia such as E. coli MWEC101-b (KR8903681), E. coli NRRL B-12141, NRRL B-12145, NRRL B_l 2146 and NRRL B-12147 (US Pat.No. 4,407,952). Les, not limited to these. In addition, E. coli K-12 [W3110 (tyrA) / pPHAB] (FERM BP_3566), E. coli K- that retains the gene encoding chorismate mutase-prefenate dehydratase with feedback inhibition released 12 [W3110 (tyr A) / pPHAD] (FERM BP_12659), E. coli K-12 [W3110 (tyrA) / pPHATerm] (FERM B P-12662) and E. coli K-12 named AJ 12604 [ W3110 (tyrA) / pBR-aroG4, pAC MAB] (FERM BP-3579) can also be used (EP 488424 Bl). Furthermore, L-hue belonging to the genus Escherichia with increased activity of the protein encoded by the yedA gene or the ydd G gene. Dialanin-producing bacteria can also be used (US Patent Application Publications 2003/0148473 A1 and 2003/01 57667 Al, WO03 / 044192).

[0059] L Tryptophan producing bacteria

Examples of L-trybutophane-producing bacteria or parent strains for inducing them include E. coli JP4735 / PMU3028 (DSM10122) and JP6015 / pMU91 (deficient in the tryptophanyl-tRNA synthetase encoded by the mutant t 卬 S gene. (DSM10123) (U.S. Pat.No. 5,756,345), serA allele encoding phosphoglycerate dehydrogenase not subject to feedback inhibition by serine and tn ^ E allele encoding anthranilate synthase not subject to feedback inhibition by tryptophan. E. coli SV164 (pGH5) (US Pat. No. 6,180,373), E. coli AGX17 (pGX44) (NRRL B-12263) and AGX6 (pGX50) aroP (NRRL B-12264) deficient in tryptophanase ) (U.S. Pat.No. 4,371,614) and E. coli AGX17 / pGX50, pACKG4-pps (WO970833 3, U.S. Pat.No. 6,319,696) with increased phosphoenolpyruvate production capacity. Force include strains belonging to the A genus S, limitation to such les. L-trifutophan-producing bacteria belonging to the genus Escherichia with increased activity of the protein encoded by the yedA gene or the yddG gene can also be used (US Patent Application Publications 2003/0148473 A1 and 2003/0157667 Al).

[0060] Examples of L-trybutophane-producing bacteria or parent strains for inducing them include anthranilate synthase (t 卬 E), phosphoglycerate dehydrogenase (serA), 3-deoxy D arabino hepturonic acid Ί phosphate synthase (aroG), 3 dehydroquinate synthase (aroB), shikimate dehydrogenase (aroE), shikimate kinase (aroL), 5-enol pyruvylshikimate 3-phosphate synthase (aroA), chorismate synthase (aroC) Also, a strain in which the activity of one or more enzymes selected from prephenate dehydratase, chorismate mutase, and tryptophan synthase (t 卬 AB) is enhanced is also included. Presulfate dehydratase and chorismate mutase are encoded by the pheA gene as a bifunctional enzyme (CM-PD). Among these enzymes, phosphoglycerate dehydrogenase, 3-deoxy-1D-alapinohepronophosphate-1_phosphate synthase, 3_dehydroquinate synthase, shikimate dehydratase, shikimate kinase, 5-benoleic acid pyruvyl shikimate 3_phosphate synthase, chorismate synthase, prephosphate dehydratase, coli Smutate mutase-prefenate dehydrogenase is particularly preferred. Since both anthranilate synthase and phosphoglycerate dehydrogenase are subject to feedback inhibition by L-tryptophan and L-serine, mutations that cancel the feedback inhibition may be introduced into these enzymes. Specific examples of strains having such mutations include E. coli SV164 carrying a desensitized anthranilate synthase and a mutant serA gene encoding phosphoglycerate dehydrogenase from which feedback inhibition has been released. And a transformant obtained by introducing plasmid pGH5 (WO 94/08031) into E. coli SV164.

[0061] Examples of L_trybutophane-producing bacteria or parent strains for deriving the same include strains into which a tryptophan operon containing a gene encoding an inhibitory anthranilate synthase has been introduced (Japanese Patent Laid-Open No. 57-71397) JP, 62-244382, U.S. Pat. No. 4,371,614). Furthermore, L-tryptophan-producing ability may be imparted by increasing the expression of a gene encoding tryptophan synthase in the tryptophan operon (t 卬 BA). Tryptophan synthase consists of α and β subunits encoded by t 卬 A and t 卬 B genes, respectively. Furthermore, L-tryptophan production ability may be improved by increasing the expression of isocitrate triase-malate synthase operon (WO2005 / 103275)

[0062] L proline-producing bacteria

Examples of L proline-producing bacteria or parent strains for inducing them include strains belonging to the genus Escherichia, such as Ε · coli 702ilvA (VKPM B-8012) (EP 1172433), which lacks the ilvA gene and can produce L proline. The powers mentioned are not limited to these.

[0063] The bacterium used in the present invention may be improved by increasing the expression of one or more genes involved in L_proline biosynthesis. An example of a gene preferable for L-proline-producing bacteria includes a proB gene (German Patent No. 3127361) that codes for gnoretamate kinase that has been desensitized to feedback inhibition by L-proline. Furthermore, the bacteria used in the present invention may be improved by increasing the expression of one or more genes encoding proteins that excrete L one amino acid from bacterial cells. Examples of such genes include b2682 gene and b2683 gene (ygaZH gene) (EP1239041 A2). [0064] Examples of bacteria belonging to the genus Escherichia having L-proline producing ability include NRRL B-12 403 and NRRL B-12404 (British Patent No. 2075056), VKPM B-8012 (Russian Patent Application 2 000124295), Examples include E. coli strains such as plasmid mutants described in German Patent No. 3127361 and plasmid mutants described in Bloom FR et al (The 15th Miami winter symposium, 1983, p. 34).

[0065] L_Arginine producing bacteria

Examples of L-arginine-producing bacteria or parent strains for inducing them include E. coli 237 strain (VKPM B-7925) (US Patent Application Publication 2002/058315 Al) and mutant N-acetylidanolate synthase. Arginine production in which the argA gene coding for N-acetyl dartamate synthetase is introduced, the derivative strain (Russian patent application 2001112869), E. coli 382 strain (VKPM B-7926) (EP1170358A1) Examples include, but are not limited to, strains belonging to the genus Escherichia such as a strain (EP1170361A1).

[0066] Examples of L-arginine-producing bacteria or parent strains for deriving the same also include strains in which expression of one or more genes encoding L-arginine biosynthetic enzymes are increased. Examples of such genes include the N-acetyl glutamyl phosphate reductase gene (argC), the ornitine acetyl transferase gene (argj), the N_acetyl daltamate kinase gene (argB), the acetyl olnitine transaminase gene ( argD), ornithine rubamoyltransferase gene (argF), arginosuccinate synthetase gene (argG), arginosuccinate lyase gene (argH), and rubamoyl phosphate synthetase gene (carAB).

[0067] L-valine producing bacteria

Examples of L-valine-producing bacteria or parent strains for inducing them include, but are not limited to, strains modified to overexpress the ilvGMEDA operator (US Pat. No. 5,998,178). Nare ,. It is preferable to remove the region of the ilvGMEDA operon required for attenuation so that the operon expression is not attenuated by the L-parin produced. Furthermore, it is preferable that the ilvA gene of the operon is disrupted to reduce the threonine deaminase activity.

Examples of L-valine-producing bacteria or parent strains for inducing them include aminoacyl t_RNA Examples include mutants having a synthetase mutation (US Pat. No. 5,658,766). For example, E. coli VL1970 having a mutation in the ileS gene encoding isoleucine tRNA synthetase can be used. E. coli VL1970 was issued on 24 June 1988 in Lucian 'National' Collection 'Ob'Industrial'Microorganisms (VKPM) (1 Dorozhny proezd., 1 Moscow 117545, Russia) with accession number VKPM B -Deposited at 4411.

Furthermore, a mutant strain (WO96 / 06926) that requires lipoic acid for growth and / or lacks H ⁺ -ATPase can be used as a parent strain.

[0068]: L-isoleucine-producing bacterium

Examples of L-isoleucine-producing bacteria or parent strains for deriving them include mutant strains resistant to 6-dimethyloleaminobrin (Japanese Patent Laid-Open No. 5-304969), thiisoleucine, iso-orthoisine hydroxamate, etc. Mutants that are resistant to isoleucine analogs, as well as mutants that are resistant to DL-ethionine and Z or arginine hydroxamate (JP-A-5-130882). ,. Furthermore, a recombinant strain transformed with a gene encoding a protein involved in L-isoleucine biosynthesis such as threonine deaminase and acetohydroxy acid synthase can also be used as a parent strain (JP-A-2-458, FR 0356739, and US Pat. No. 5,998,178).

[0069] When breeding the above L-amino acid-producing bacterium by genetic recombination, the gene used is not limited to the gene having the above-described gene information or a gene having a known sequence, and the function of the encoded protein is not limited. As long as it is not impaired, a gene having a conservative mutation such as a homologue or artificially modified gene of the gene can also be used. That is, a gene encoding a protein having a sequence containing a substitution, deletion, insertion or addition of one or several amino acids at one or several positions in the amino acid sequence of a known protein. There may be.

[0070] Here, "one or several" differs depending on the position of the amino acid residue in the three-dimensional structure of the protein and the type of amino acid residue, but specifically, preferably 1 to 20, more preferably. Means 1-10, more preferably 1-5. Conservative mutations are polar between Phe, Trp, and Tyr when the substitution site is an aromatic amino acid, and between Leu, Ile, and Val when the substitution site is a hydrophobic amino acid. In the case of an amino acid, a salt between Gln and Asn When it is a basic amino acid, it is placed between Lys, Arg, and His, when it is an acidic amino acid, it is between Asp and Glu, and when it is an amino acid having a hydroxyl group, it is placed between Ser and Thr. Mutation. Representative conservative mutations are conservative substitutions. Specifically, substitutions considered as conservative substitutions include substitution from Ala to Ser or Thr, Arg to Gln, His or Lys. Substitution from Asn to Glu, Gln, Lys, His or Asp, Asp force, et al. From Asn, Glu or Gin, Cys to Ser or Ala, Gin to Asn, Glu, Lys, His, Asp Or Arg, Glu to Gly, Asn, Gln, Lys or Asp, Gly to Pro, His to Asn, Lys, Gln, Arg or Tyr, lie force, et al. Leu, Met, Val or Phe substitution, Leu to Ile, Met, Val or Phe substitution, Lys to Asn, Glu, Gln, His or Arg, Met to Ile, Leu, Val or Phe, Phe to TYp, Tyr, Met, lie or Leu substitution, Ser to Thr or Ala substitution, Thr to Ser or Ala substitution, TYp to Phe or Tyr substitution, Tyr to Hi Substitution to s, Phe or TYp, and substitution to Val force, et al. Met, lie or Leu. In addition, amino acid substitutions, deletions, insertions, attachments, or inversions as described above include naturally occurring mutations (mutant or mutants) based on individual differences or species differences of the microorganism from which the gene is derived. including those produced by variants). Such a gene can be generated, for example, by site-directed mutagenesis so that the amino acid residues at specific sites of the encoded protein include substitutions, deletions, insertions or additions. Can be obtained by modifying.

[0071] Further, a gene having a conservative mutation as described above is 80% or more, preferably 90% or more, more preferably 95% or more, particularly preferably 97% or more, based on the entire encoded amino acid sequence. And a gene that encodes a protein having the same homology and having a function equivalent to that of the wild-type protein.

In addition, each codon in the gene sequence can be used in the host into which the gene is introduced, or can be replaced with a codon.

[0072] A gene having a conservative mutation may be obtained by a method usually used for mutation treatment, such as treatment with a mutation agent.

[0073] In addition, a gene is hybridized under a stringent condition with a complementary sequence of a known gene sequence or a probe that can be prepared from the complementary sequence, and is equivalent to a known gene product. It may be a DNA encoding a protein having the following functions. Here, “stringent conditions” refers to conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed. For example, DNAs with high homology, for example, 80% or more, preferably 90% or more, more preferably 95% or more, particularly preferably 97% or more, are hybridized to each other. This is a condition under which DNAs with lower homology do not hybridize with each other, or under normal Southern hybridization 60 conditions. C, IX SSC, 0.1% SDS, preferably 0.1 X SSC, 0.1% SDS, more preferably 68. The conditions include washing once, more preferably 2 to 3 times at a salt concentration and temperature corresponding to C, 0.1 X SSC and 0.1% SDS.

[0074] As the probe, a part of the complementary sequence of the gene can also be used. Such a probe can be prepared by PCR using an oligonucleotide prepared on the basis of a known gene sequence as a primer and a DNA fragment containing these base sequences as a cage. For example, when a DNA fragment having a length of about 300 bp is used as a probe, the hybridization washing conditions include 50 ° C., 2 × SSC, and 0.1% SDS.

[0075] <3> L Amino Acid Production Method

In the method for producing L-amino acid of the present invention, a bacterium that belongs to the family Enterobacteriaceae and has the ability to produce L-amino acid is cultured in a medium containing glycerol as a carbon source, and L-amino acid is produced and accumulated in the culture. Collect L-amino acids from the culture.

[0076] Glycerol to be used may be used at any concentration that is suitable for producing L-amino acid. When used as a single carbon source in the medium, it is preferably about 0. lw / v% to 50 w / v%, more preferably about 0.5 w / v% to 40 w / v%, particularly preferably lwZv% to 30 w / About v% is contained in the medium. Glycerol can also be used in combination with other carbon sources such as dalcose, fructose, sucrose, molasses and starch hydrolysates. In this case, glycerol and other carbon sources can be mixed in any ratio S, the ratio of glycerol in the carbon source is 10 wt% or more, more preferably 50 wt% or more, more preferably Is preferably 70% by weight. Other preferred carbon sources are glucose, fructose, sucrose, ratatoose, galactose, molasses, starch hydrolysates, sugars obtained by hydrolysis of biomass, Alcohols such as tanol, and organic acids such as fumaric acid, succinic acid, and succinic acid. Of these, the gnole course is preferred. Also particularly preferred is a mixture comprising crude glycerol and glycolose in a weight ratio of 50:50 to 90:10.

The preferred concentration and initial concentration of glycerol at the start of culture are as described above, but glycerol may be added depending on the consumption of glycerol during the culture.

[0077] A preferable medium in the present invention is a medium supplemented with crude glycerol. When using crude glycerol, the crude glycerol may be added to the medium so that the concentration of glycerol is the above concentration, depending on the purity of glycerol.

Further, both glycerol and crude glycerol may be added to the medium.

[0078] As a medium to be used, a medium conventionally used in the fermentation production of L_amino acids using microorganisms can be used. That is, in addition to a carbon source, a normal medium containing a nitrogen source, inorganic ions, and other organic components as required can be used. Here, as the nitrogen source, inorganic ammonium salts such as ammonium sulfate, ammonium chloride and ammonium phosphate, organic nitrogen such as soybean hydrolysate, ammonia gas, aqueous ammonia and the like can be used. Organic micronutrient sources include vitamin B, L-homoserine, etc.

1

It is desirable to contain an appropriate amount of the required substance or yeast extract. In addition to these, small amounts of potassium phosphate, magnesium sulfate, iron ions, manganese ions, etc. are added as necessary. The medium used in the present invention may be either a natural medium or a synthetic medium as long as it contains a carbon source, a nitrogen source, inorganic ions, and other organic trace components as required.

[0079] Cultivation should be carried out under aerobic conditions: for ~ 7 days, the culture temperature should be 24 ° C-45 ° C, and the pH during cultivation should be 5-9. For pH adjustment, inorganic or organic acidic or alkaline substances, ammonia gas, etc. can be used. L-amino acids can be recovered from the culture medium by combining an ion exchange resin method, a precipitation method, and other known methods. When L-amino acid accumulates in the microbial cells, for example, the microbial cells are disrupted by ultrasonic waves, and the microbial cells are removed by centrifugation. Amino acids can be recovered.

Example Hereinafter, the present invention will be described in more detail with reference to examples. In the examples, reagent grade grade (manufactured by Nacalai Testa Co., Ltd.) was used as the reagent glycerol, and crude glycerol (GLYREX, Nowit DCA_F, and R Glycer in) produced during the biodiesel production process was used as the crude glycerol. The glycerol purity of this crude glycerol was 86% by weight for crude glycerol GLYREX, 79% by weight for crude glycerol Nowit DCA-F, and 78% by weight for crude glycerol R Glycerin.

GLYREX is manufactured by FOX PETROLI Italy (FOX PETROLI SPA Sede legale e uffici, vi a Senigallia 29, 61100 Pesaro), from SVG Italy (SVG ITALIA SrL Via A. Majani, 2, 40122 Bologna (BO)) Crude Daly Seronore, sold as an animal feed additive, was obtained. Nowit DCA—F is available from Nordische Oelwerke Walther Carrouxy (Nordische Oelwerke Walther Carroux GmbH & Co KG, Postfach 930247 Industri estrasse 61-65, 21107 Hamburg). R Glycerin is a crude glycerol sold by the German company Inter-Harz (Inter-Harz GmbH Postfach 1411 Rostock- Koppel 17, 25314 Elmshom, 25365 Kl. Oifenseth-Sparrieshoop).

[Example 1] Growth of wild strain in minimal medium

Escherichia coli MG1655 strain was cultured on LB agar medium (tryptone 10 g, yeast extract 5 g / L, NaC 1 10 g / L, agar 15 g / L) at 37 ° C for 16 hours, cells were scraped with ase, Suspended in 0.9% NaC1. Add 5 ml of M9 medium (Na HPO 7Η · 12.8 g / L, K HPO 0.6 g, NaCl, containing 0.4% (w / v) glucose, reagent glycerol or crude glycerol as carbon source.

2 4 2 2 4

0.5 g / L, NH CI lg, 2 mM MgSO, O. lmM CaCl), and tested at 37 ° C for 24 hours.

4 4 2

Culture was performed in a test tube.

[0082] This culture broth was diluted, spread on an LB agar medium, and cultured at 37 ° C for 16 hours. In order to accurately measure the degree of growth, the number of grown colonies was counted as the number of viable bacteria. Table 1 shows the average value of the results of culturing in two test tubes.

[0083] [Table 1]

-table 1

Carbon source Viable count (per ml)

Glucose 1.0 X 10 ⁵

Reagent glycerol 1. 2 10 ⁵

Crude glycerol GLYREX 6. 6 10 ⁶ [0084] The reagent glycerol showed growth equivalent to or better than that of gnolecose, and the crude glycerol showed unexpectedly good growth of 50 times or more than that of glucose.

[Example 2] L-threonine production culture

[0085] Escherichia coli VKPM B-5318, an L-threonine-producing bacterium, was added to a LB agar medium (tryptone 10 g / L, yeast extract 5 g / re NaCl 10 g / L, agar 15 g / L L) and cultured at 37 ° C for 24 hours. The cells on the agar medium are scraped, inoculated into a 500 ml volumetric flask containing 20 mL of L-threonine production medium containing 20 mg / L of streptomycin sulfate, and cultured at 40 ° C for 24 hours. went. The main culture was carried out in a medium using glucose, reagent glycerol and crude glycerol alone as a carbon source, and a medium using glucose and reagent glycerol 1: 1 or glucose and crude glycerol 1: 1. . The total amount of carbon source was adjusted to 40 g / L.

[0086] (Composition of L-threonine production medium)

(A ward)

Carbon source 40g / L

MgSO 7Η 0 lg / L

4 2

(Nada Ward)

Yeast extract 2g / L

FeSO 7Η 0 lOmg / L

4 2

MnSO 4Η 0 lOmg / L

4 2

Lg ΡΟ lg / L

twenty four

(ΝΗ) SO 16g / L

4 2 4

(C ward)

Calcium carbonate 30g / L

Autoclave sterilization at 115 ° C for 10 minutes in districts A and B, 180 ° C for 3 hours in zone C Dry heat sterilization After cooling to room temperature, mix the three.

After completion of the culture, consumption of the added sugar and glycerol was confirmed with BF-5 (Oji Scientific Instruments), and the degree of growth was measured by turbidity (OD) at 600 nm. The amount of L-threonine was measured by liquid chromatography. Table 2 shows the average value of the culture results of two flasks. Shown in

Under the main culture conditions, the amount of L-threonine when glucose was used as the carbon source remained low, but when the reagent glycerol was mixed or added alone, the amount of L-threonine was significantly improved. Furthermore, when crude glycerin was used alone, a greater increase in the amount of L-threonine was observed than when reagent glycerin was used alone.

[Table 2] Table 2

Element ^ 0D Thr (g / l) Glucose 40 g / l 9.6 3.8 Glucose 20 g / l + Reagent glycerol 20 g / l 11.6 12.3 Reagent glycerol 40 g / l 10.1 9.9 Glucose 20 g / l + Crude glycerol GLYREX 20 g / l 11.2 12.5 Crude glycerol GLYREX 40g / l 10.4 12.0

[Example 3] L-lysine production culture

Escherichia coli WC196DcadADldc / pCABD2 (this strain is referred to as “WC196LC / pCABD2”) described in WO 2006/078039 pamphlet was used as an L-lysine-producing bacterium. Escherichia coli WC196LC / pCABD2 was cultured at 37 ° C for 24 hours on LB agar medium (trypton 10 g / L, yeast extract 5 g / L, NaCl 10 g / L agar 15 g / L) containing streptomycin sulfate 20 mg / L did. The cells on the agar medium are scraped off, inoculated into a 500 ml volumetric flask containing 20 mL of L-lysine production medium containing 20 mg / L of streptomycin sulfate, and cultured at 37 ° C for 48 hours. went. The main culture consists of a medium using glucose, reagent daricerol, and crude glycerol alone as a carbon source, and a medium using glucose and reagent glycerol 1: 1, or glucose and crude glycerol 1: 1. It carried out in. The total carbon source amount was set at 40g / L.

[0090] (L-lysine production medium composition)

(A ward)

Carbon source 40g / L

(B ward)

Yeast extract 2g / L

FeSO 7Η O 10mg / L MnSO 4Η O lOmg / L

KH PO lg / L

(NH) SO 24g / L

(C ward)

Calcium carbonate 30g / L

[0091] After completion of the culture, consumption of the added sugar and glycerol was confirmed with BF-5 (Oji Scientific Instruments), and the degree of growth was measured by turbidity (OD) at 600 mm. The amount of L-lysine was measured by Biotech Analyzer AS210 (Sakura Seiki). Table 3 shows the average value of the results of culturing two flasks at a time.

When the reagent glycerol was mixed or added alone, the amount of L-lysine significantly decreased compared to the amount of L-lysine when gnolecose was used as the carbon source. However, when crude glycerin was mixed or used alone, an increase in the amount of L-lysine was observed as compared with the case of using reagent glycerin, and an amount comparable to the amount of L-lysine when gnolecose was used as a carbon source could be obtained. done.

[0092] [Table 3] Carbon source OD Lys (g / l) Glucose 40 g / l 8. 6 14.9 Glucose 20 g / l + Reagent glycerol 20 g / l 10. 4 13.3 Reagent glycerol 40 g / l 10. 0 13.4 Glucose 20g / l + Crude Glycerol GLYREX 20g / l 10.7 14.3 Crude Glycerol GLYREX 40g / l 9. 9 14.5

[Example 4] L-lysine production culture with various crude glycerol

L lysine-producing bacterium Escherichia coli WC196LC / pCABD2 on LB agar medium (trypton 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, agar 15 g / L) containing streptomycin sulfate 20 mg / L The cells were cultured at 37 ° C for 24 hours. Cells on the agar medium are scraped, inoculated into a 500 ml Sakaguchi flask containing 20 mL of L-lysine production medium containing 20 mg / L of streptomycin sulfate, and cultured for 48 hours at a culture temperature of 37 ° C. went. In the main culture, GLYREX, Nowit DCA_F, Alternatively, R Glycerin was used. The total carbon source amount was 40 g / L for all.

[0094] After completion of the culture, consumption of the added glucose and glycerol was confirmed with BF-5 (Oji Scientific Instruments), and the degree of growth was measured by turbidity (OD) at 600 nm. The amount of L-lysine was measured with a Biotech Analyzer AS210 (Sakura Seiki). Table 4 shows the average value of the results of culturing two flasks at a time.

GLYREX, Nowit DCA- for the amount of L-lysine when the reagent glycerol is used as the carbon source

The amount of L-lysine increased when either F or R Glycerin or crude glycerol was used.

[0095] [Table 4]

Table 4

Carbon source OD Lys (g / l) Reagent glycerol 40g / l 9. 7 14.9 Crude glycerol GLYREX 40g / l 9. 8 16.6 Crude glycerol Now it DCA-F 40g / l 10. 0 15. 9 Crude glycerol RG l ycer in 40g / l 9. 9 16. 4

[0096] [Example 5] Construction of L-glutamic acid-producing bacterium of Pantoea ananatis

The gltA gene of plasmid RSFCPG (see European Patent Publication No. 1233068) carrying the citrate synthase gene (gltA), phosphoenolpyruvate carboxylase gene (ppc), and glutamate dehydrogenase gene (gdhA) derived from Escherichia coli, Plasmid RSFPPG was constructed by replacing the methyl succinate synthase gene of Escherichia coli (p 卬 C) (WO 2006/0516 60 pamphlet).

[0097] Primer 1 (SEQ ID NO: 1) and primer 2 (SEQ ID NO: 2) were designed to amplify the RSFCPG gltA gene other than the ORF. Using this primer, PCR was carried out using RSFCPG in a saddle shape to obtain a fragment of about 14.9 kb. On the other hand, the methyltaenoic acid synthase gene (p 卬 C) derived from Escherichia coli was used as primer 3 (SEQ ID NO: 3) and primer 4 (SEQ ID NO: 4), and the chromosomal DNA of Escherichia coli strain W3110 was used as a saddle type. PCR was performed, and a fragment of about 1.2 kb was obtained. Both PCR products were treated with BglII and Kpnl, and after ligation, Escherichia coli JM109 strain was transformed. All the emerged colonies were collected, and the plasmid was extracted as a mixture. This plasmid mixture transforms the citrate synthase (CS) -deficient strain E. coli strain 8330, containing 50 mg / L uracil, 5 mg / L thiamine-HC1 M9 minimal medium (Na HPO · 7Η 2.8 12.8 g / L, Κ HPO 0.6 g, NaCl 0.5 g / L, NH C

2 4 2 2 4 4

1 lg / L, 2 mM MgSO, O.lmM CaCl 3). Extract the plasmid from the emerging strain,

4 2

This was called RSFPPG. The plasmid RSFPPG was introduced into Pantoea ananatis NP106 strain, which is an L-glutamic acid-producing bacterium, to construct an L-glutamic acid-producing bacterium NP106 / RSFPPG (this strain is referred to as “NA1 strain”).

[0098] The NP106 strain was obtained as follows. Pantoea's Ananatis AJ1 3601 strain exemplified above was cultured overnight in LBGM9 liquid medium at 34 ° C, diluted to 100-200 colonies per plate, and LBGM9 plate containing tetracycline 12.5mg / L We applied to. The colonies that emerged were replicated on an LBGM9 plate containing tetracycline 12.5 mg / re and chloramphenicol 25 mg / L, and a strain that became chloramphenicol-sensitive was selected, and a strain from which pSTVCB had dropped was obtained and named G106S. . Furthermore, the G106S strain was cultured with shaking in LBGM9 liquid medium at 34 ° C overnight, diluted to 100 to 200 colonies per plate, and applied to an LBGM9 plate containing no drug. The colonies that appeared were replicated on LBGM9 plates containing 12.5 mg / L of tetracycline and LBGM9 plates containing no drug, and the strains that became tetracycline-sensitive were selected. A strain in which RSFCPG had dropped was obtained and named NP106. The NP106 strain thus obtained is a strain that does not have both plasmids RSFCPG and pSTVCB carried by the AJ13601 strain.

[Example 6] L-glutamic acid production culture

L Pantoea's Ananatis NA1 strain, a gnoretamic acid-producing bacterium, was added to LBGM9 agar medium (tetraprine hydrochloride 12.5 mg / L, tryptone 10 g, yeast extract 5 g / L, NaCl 10 g, Na HPO 7O O 12.8 g K HPO 0.6 g /, NaCl 0.5 g / L, NH CI lg / L,

2 4 2 2 4 4 Lucose 5g / l, agar 15g / L) was cultured at 34 ° C for 24 hours. The cells on the agar medium are scraped off, inoculated into a test tube into which 5 mL of L-glutamic acid production medium containing tetracycline hydrochloride 12.5 mg / L has been injected, and cultured at a culture temperature of 34 ° C for 24 hours. It was. In the main culture, sucrose, glucose, reagent glycerol, and crude glycerol GLYREX were used as carbon sources. The total carbon source amount was 30 g / L.

[0100] (L-Glutamic acid production medium composition)

(A ward) Carbon source 30g / L

MgSO7Η O 0.5g / L

4 2

(B ward)

(NH) SO 20g / L

4 2 4

KH PO 2g / L

twenty four

FeSO -7H O 20mg / L

4 2

MnSO -5H O 20mg / L

4 2

Yeast extract 2g / L

Calcium pantothenate 18mg / L

(C ward)

Calcium carbonate 20g / L

[0101] After completion of the culture, the degree of growth was measured by turbidity (OD) at 600 nm, and consumption of the added glucose and glycerol was confirmed with BF_5 (Oji Scientific Instruments). The amount of sucrose and L-daltamic acid was measured with Biotech Analyzer AS210 (Sakura Seiki). Table 5 shows the average value of the results of culturing in two test tubes.

When the reagent glycerol was used, the amount of L-glutamic acid was increased compared to the amount of L-glutamic acid when gnolecose was used as the carbon source. Furthermore, when crude glycerol GLYR EX is used, the amount of L-glutamic acid is significantly higher than when glucose or reagent glycerol is used, and a higher amount of L-glutamic acid is obtained than when sucrose is used. I was able to.

[0102] [Table 5] Table 5

Carbon source 0D Glu (g / l) Sucrose 40 g / l 12.4 16.8 Glucose 40 g / l 13.6 14.1 Reagent glycerol 40 g / l 14.0 14.9 Crude glycerol GLYREX 40 g / l 13.6 17.7 [Example 7] Component analysis of crude glycerol

Component analysis of each crude glycerol of GLYREX, Nowit DCA_F, and R Glycerin was performed. As for the measurement method, glycerol and methanol were measured by gas chromatography. Total nitrogen was measured by the Kjeldahl method, and ether solubles were measured by the Soxhlet extraction method. Formic acid and acetic acid were measured by high-performance liquid chromatography, and chloride ion and sulfate ion were measured by ion chromatography. Sodium, potassium and copper were measured by atomic absorption spectrophotometry, and phosphorus, iron, calcium, magnesium, manganese and zinc were measured by ICP (Inductively Coupled Plasma) emission spectrometry. Table 6 shows the measurement results of the content (g) per 100 g.

[0104] [Table 6] Table 6

Measurement item GLYREX No it DCA-F RG l cer in glycerol 85. 9 78. 5 78. 2 Total nitrogen at 0. 01 0. 01 0 0. 01 Ether soluble 0. 1 0. 3 <0. 1

Na 0. 16 2. 09 2. 1 1

K 2. 45 0. 0062 0. 144 ci- 2. 33 2. 62 3. 38

S0 ₄ ² "<0. 05 0. 06 <0. 05 Methanol 0. 0054 0. 1 1 0. 0015 Formic acid 0. 02 0. 01 0. 01 Acetic acid 0. 03 0. 02 0. 03

P 0. 0085 0. 0787 0. 0219

Mg 0. 001 0. 0003 0. 0003

Fe 0. 0036 0. 00043 0. 00054

Ca 0. 0032 0. 001 1 0. 001

Mn 0. 00003 0. 00001 0. 00001

Cu 0. 00002 0. 00008 <0. 00001

Zn 0. 00077 <0. 00001 0. 00001

Na + K + G + SO / "* 4. 94 4. 7762 5. 63

Mg + Fe + Ca * 0. 0078 0. 00183 0. 00084

Mn + Cu + Zn * 0. 00082 0. 00009 0. 00001

*: Calculated with 0 below the measurement limit

[Explanation of Sequence Listing]

SEQ ID NO: 1: Primer for amplifying the gltA gene other than the ORF

SEQ ID NO: 2: Primer for amplifying the non-ORF part of gltA gene

SEQ ID NO: 3: Primer for prpC gene amplification SEQ ID NO: 4: Primer for prpC gene amplification

Industrial applicability

According to the present invention, it is possible to produce L_amino acid at low cost by using a new inexpensive carbon source.

Claims

The scope of the claims

[I] Bacteria belonging to the family Enterobacteriaceae and having L_amino acid-producing ability are cultured in a medium containing glycerol as a carbon source, and L-amino acid is produced and accumulated in the culture. A method for producing an L-amino acid characterized by collecting an acid.

[2] The method according to claim 1, wherein the initial concentration of glycerol in the medium is 1 to 30 w / v%.

[3] The method according to claim 1 or 2, wherein the medium is a medium supplemented with crude glycerol.

[4] The method according to claim 3, wherein the crude glycerol is crude glycerol produced in production of biodiesel fuel.

[5] The method according to claim 3 or 4, wherein the crude glycerol power is glycerol capable of producing more L-amino acids as compared with the reagent glycerol when used as a carbon source.

6. The method according to any one of claims 1 to 5, wherein the bacterium belongs to the genus Escherichia.

7. The method according to any one of claims 1 to 5, wherein the bacterium belongs to the genus Pantoea.

8. The method according to claim 6, wherein the bacterium is Escherichia coli.

[9] The method according to any one of claims 1 to 8, wherein the L-amino acid is one or more L_amino acids selected from the group consisting of L-threonine, L-gnoretamic acid, and L-lysine. .

[10] The L-amino acid is L-threonine, and the bacterium is aspartate semialdehyde dehydrogenase, asphalt kinase I encoded by the thr operon, homoserine kinase, aspartate aminotransferase, and threonine synthase 10. The method according to claim 9, wherein the activity of one or more enzymes selected from the group consisting of is enhanced.

[II] The L_amino acid is L_lysine, and the bacterium is dihydrodipicolinate reductase, diaminopimelate decarboxylase, diaminopimelate dehydrogenase, phosphoenolpyruvate carboxylase, spartate aminotransferase, diaminopimelate epimerase, and aspartate. Consists of tate semialdehyde dehydrogenase, tetrahydrodipicolinate succinylase, and succinyldiaminopimelate deacylase 10. The method according to claim 9, wherein the activity of one or more enzymes selected from the group is enhanced and / or the activity of lysine decarboxylase is attenuated.

[12] The L amino acid is L-gnoretamic acid, and the bacterium is selected from the group consisting of glutamate dehydrogenase, citrate synthase, phosphoenolpyruvate carboxylase, and methyl citrate synthase. The method according to claim 9, wherein the activity of the ketognoletalate dehydrogenase is attenuated.

[13] The L-amino acid is L-tryptophan, and the bacterium is phosphoglycerate dehydrogenase, 3-deoxy-1D-alapinoheplonate monophosphate 7_phosphate synthase, 3_ dehydroquinate synthase, shikimate dehydrogenase , Shikimate kinase, 5-enol pyruvyl shikimate 3-phosphate synthase, chorismate synthase, prefenate dehydratase, chorismate mutase 10. The method of claim 9, wherein